The objectives of this research program are to elucidate the pathway of ubiquinone biosynthesis in animal cells with emphasis on (a) the identity of the intermediates, (b) the nature of the enzymes involved in each step, including the mechanism of the reaction, and (c) the factors which regulate the pathway under various conditions. It has been demonstrated that ubiquinone-9 biosynthesis can occur in rat liver mitochondria and hence efforts will be made to solubilize given enzymes in the biosynthetic sequence from this source. 2-Nona-prenyl-6- methoxyphenol and 5-demethoxyubiquinone-9 have been isolated from rat liver and identified as intermediates in the biosynthesis of ubiquinone- 9 in the rat. These and other compounds (p-hydroxybenzoyl-CoA, 4- hydroxy-3-nonaprenyl-benzoyl-CoA, 5 hydroxy-2-nonaprenyl-3-methyl-6- methoxy-1,4-benzoquinone) will be synthesized chemically or biochemically with isotopic labels as substrates for the study of the alkylation, decarboxylation, hydroxylation, and methylation reactions which characterize ubiquinone biosynthesis. S-adenosyl-methionine- methyl-C14 will be employed as methyl donor in the latter studies. The regulatory function of polyprenyl-pyrophosphates which are synthesized in the cytoplasm will also be studied.